Studies on the mechanism of action of merbarone (5-[5-phenylcarboxamidol]-2-thiobarbituric acid) have been extended to include the toxicity of this novel oncolytic agent towards human as well as rodent cells. Merbarone is cytocidal to several lung tumor lines propagated in continuous culture, as it is towards murine lymphoblasts, but the median inhibitory concentrations of the drug exhibit a significant spread, ranging from 9 MuM in the case of NCI-H358 to 55 MuM in the case of NCI-H322. Versus L1210 cells, a strain previously shown to exhibit exquisite sensitivity to merbarone both in vivo and in vitro, the drug produces a powerful time-dependent arrest of DNA synthesis and is accompanied, in human lymphocytes, by chromosomal abnormalities. When L1210 cells are exposed to an IC50 concentration of the drug (15 MuM) for 16 hours, prominent single strand breaks in their DNA are demonstrable by alkaline elution analyses. These breaks reseal at a rate some fifty times slower than those produced by Gamma-irradiation. Merbarone also engenders peroxidation of rat liver microsomal lipids in the presence of NADPH and oxygen. On a molar basis this effect is greater than that produced by adriamycin - an oncolytic drug well known for its ability to generate toxic free radicals. However, in contrast to the case with adriamycin, superoxide dismutase did not influence the peroxidation caused by merbarone whereas the antioxidant butylated hydroxyanisole was a potent antagonist of this activity. ESR analyses of microsomes incubated aerobially with merbarone in the presence of NADPH and spin-trapping reagents demonstrated that the drug supported the production of oxygen-centered free radicals and it is hypothesized that these species are responsible both for the DNA strand breaks and lipid peroxidation which result from exposure to merbarone.